March 2005

Notes

Biol. Pharm. Bull. 28(3) 549—552 (2005)

549

Inhibitory Effects of Amino-Acid Fluids on Drug Binding to Site II of
Human Serum Albumin in Vitro
Keishi YAMASAKI,*,a Noriyuki KUGA,b Norito TAKAMURA,c Yumiko FURUYA,a Muneaki HIDAKA,aTomomi IWAKIRI,a Ryuuichi NISHII,b Manabu OKUMURA,a Hirofumi KODAMA,a
Keiichi KAWAI,d and Kazuhiko ARIMORIaa
Department of Pharmacy, Miyazaki Medical College Hospital; b Department of Radiology, Miyazaki Medical College;
5200 Kihara, Kiyotake-cho, Miyazaki-gun 889–1692, Japan: c School of Pharmaceutical Sciences, Kyusyu University of
Health and Welfare; 1714–1 Yoshino-machi, Nobeoka 882–8508, Japan: and d School of Health Sciences, Faculty of
Medicine, Kanazawa University; 5–11–80 Kodatsuno, Kanazawa 920–0942, Japan.
Received August 23, 2004; accepted December 9, 2004; published online December 24, 2004

The effects of amino-acid fluids on ligand binding to human serum albumin (HSA) were investigated by fluorescence and ultrafiltration techniques. Warfarin and dansylsarcosine were used as the site marker fluorescenceprobes for site I and site II of HSA, respectively. Amino-acid fluids specifically decreased the fluorescence intensity induced by dansylsarcosine-HSA binding without any effects on that induced by warfarin-HSA binding. The
ultrafiltration technique clarified that the free fraction of the site II drug, diazepam, in human serum was increased in the presence of amino-acid fluids, while no effect was observed in the free fraction of the site I drug,
warfarin. The potencies of the effect on binding to site II, observed by fluorescence and ultrafiltration techniques,correlated well with the L-tryptophan contents in amino-acid fluids or with those in L-tryptophan solutions.
Based on the comparison between the effects of amino-acid fluids and L-tryptophan solutions, we confirmed that
L-tryptophan in amino-acid fluids specifically inhibits drug binding to site II of HSA.
Key words

protein binding; albumin; parenteral nutrition; binding site; amino acid; drug interaction

Human serum albumin (HSA) is a major protein component of blood plasma and plays an important role in the regulation of colloidal osmotic pressure, antioxidant capacity ofhuman plasma, and the transport of numerous endogenous
compounds such as fatty acids, hormones, toxic metabolites(e.g. bilirubin), bile acids, amino acids, and metals.1,2) The
protein also binds a wide variety of drugs,1—3) which has asignificant impact on the pharmacokinetics and pharmacological effects of these drugs.4,5) Free drug concentration canbe affected by the presence of other drugs or endogenous
compounds, or by microenvironmental changes in diseasestates. Diminished drug binding is usually the result of either
competitive displacement from the same binding site or allosteric displacement following microenvironmental changes
at the binding site. Several drugs bind with high affinity to
one of the HSA sites4,6) and these specific binding sites havebeen characterized since several decades.7—15) Sudlow etal.7,8) characterized two sites for drug binding, namely site I(also referred to as the warfarin binding site) and site II (thebenzodiazepine binding site). Sjöholm et al.9) have suggested
the existence of one more binding site (the digitoxin site).
The location of the latter site is largely unknown; however,crystallographic studies have assigned the location of sites I
and II to subdomains IIA and IIIA of HSA, respectively.3,10,11) This assignment of sites is supported by binding
studies with fragments of HSA.12—15)
Since its first description by Wilmore and Dudrick in
1968,16) parenteral nutrition has been an integral part of the
medical management for a variety of patients who are in hypermetabolic states, or suffering from neurologic disease,gastrointestinal disease, cancer, and psychiatric illness.17)
This is because the nutrients required for humans; carbohydrates, fats, amino acids, electrolytes, vitamins, and traceminerals are considered to be available for use in parenteralfeeding formulations.18,19) Despite the highly successful use∗ To whom correspondence should be addressed.

of parenteral nutrition for several years, some adverse events
have been reported, mostly regarding the errors in management of therapy using parenteral nutrition.20) Most of the patients in whom parenteral nutrition is used, are also administered some therapeutic drugs, either orally, parenterally, or
intravenously. The interaction between these drugs and theparenteral nutrition fluids may contribute to some cases of
the adverse events. However, little information is available
on the drug–parenteral nutrition fluid interaction, most notably with respect to the change in protein binding of the
drug.21—25)
This study was undertaken to evaluate the effects of parenteral nutrition fluids, especially amino-acid fluids, on protein binding to drugs in vitro. The present findings on the interaction between amino-acid fluids and drugs were well explained based on the concept of binding sites.
MATERIALS AND METHODS
Materials HSA (fraction V, fatty acid free), warfarin,
dansylsarcosine, and L-tryptophan were purchased from
Sigma Chemical Co. (St. Louis, MO, U.S.A.). [14C]Warfarin(56 mCi/mmol) and [14C]diazepam (55 mCi/mmol) were synthesized and purified by Amersham Biosciences Corp. (Piscataway, NJ, U.S.A.) and American Radiolabelled Chemical
Inc. (St. Louis, MO, U.S.A.), respectively. All other chemicals were of analytical grade. The phosphate buffer (67 mM,
pH 7.4) used was prepared with sodium phosphate dibasic
and sodium phosphate monobasic salts. All ligand molecules
were first dissolved in methanol; the final methanol concentration was less than 1% (v/v). Amino-acid fluids were obtained as follows: Aminoleban®, Kidmin®, Amiparen®,Aminofluid®, and Aminotripa® No. 1 from Otsuka Pharmaceutical Factory, Inc. (Tokushima, Japan); Proteamin 12 X
Injection® and Fulcaliq® 1 from Tanabe Seiyaku Co., Ltd.

e-mail: kcyama@fc.miyazaki-u.ac.jp© 2005 Pharmaceutical Society of Japan

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Vol. 28, No. 3

Table 1.

Amino Acid Contents in Amino-Acid Fluids Used
Contents of amino acids (g/l)

Aminoacid
L-ArL-His- L-IsoL-Leu- L-Lyfluidsa)
ginine tidine leucine cine
sine

Ab)
Bc)
Cb)
Db)
Eb)
Fb)
Gb)
Hb)

1.58
2.46
3.09
3.15
6.00
4.50
10.50
12.23

1.20
1.04
1.47
1.50
2.40
3.50
5.00
5.30

1.12
1.88
2.35
2.40
9.00
9.00
8.00
5.97

2.50
2.99
4.12
4.20
11.00
14.00
14.00
11.38

1.76
1.77
3.09
3.15
6.10
5.05
10.50
7.87

L-Me-

thionine
0.70
0.86
1.15
1.17
1.00
3.00
3.90
4.33

L-PheL-Thre- L-Tryp- L-Vanylalaonine tophan line
nine

1.87
1.71
2.06
2.10
1.00
5.00
7.00
9.74

1.30
1.06
1.68
1.71
4.50
3.50
5.70
5.04

0.26
0.35
0.59
0.60
0.70
2.50
2.00
1.87

0.90
1.99
2.35
2.40
8.40
10.00
8.00
6.90

L-As-

L-Glu-

rine

partic
acid

tamic
acid

0.44
0.93
0.88
0.90
5.00
3.00
3.00
4.67

0.76
0.11
0.29
0.30
—
1.00
1.00
2.02

1.30
0.11
0.29
0.30
—
1.00
1.00
1.02

GlycineL-Pro-

L-Se-

line

2.14
1.22
1.74
1.77
9.00
—
5.90
15.68

0.66
1.42
1.47
1.50
8.00
3.00
5.00
10.63

L-Ty-

L-Cys-

Total
free
amino
acids

L-Cys-

L-Ala-

rosine tine

tein

nine

0.07
0.11
0.15
0.15
—
0.50
0.50
0.57

0.20
0.22
0.29
0.30
0.30
1.00
1.00
—

1.24 20.00
1.90 22.15
2.35 29.41
2.40 30.00
7.50 79.90
2.50 72.05
8.00 100.00
8.21 113.62

—
—
—
—
—
—
—
0.23

a) A; PN-twin®-No. 1, B; Fulcaliq® 1, C; Aminotripa® No. 1, D; Aminofluid®, E; Aminoleban®, F; Kidmin®, G; Amiparen®, H; Proteamin 12 X Injection®. b) Fluids also
contain electrolytes and/or carbohydrates. c) Fluid also contains electrolytes, carbohydrates, and trace vitamins.

(Osaka, Japan); PN-twin®-No. 1 from Ajinomoto PharmaCo., Ltd. (Tokyo, Japan) (Table 1). Human pooled serum was
prepared from blood samples obtained from 5 healthy malesubjects (age 29.35.4 years). All subjects were withdrawn
from any medication for at least 5 d prior to blood sampling.
Concentration of albumin was determined to be 748 m M
using Roche COBAS Integra 400 plus (Roche Diagnostics,
Basel, Switzerland).
Fluorescence Measurement Steady-state fluorescencemeasurements were made using a Shimadzu RF-5300 PC
spectrofluorometer (Shimadzu Co., Kyoto, Japan) with thermostated devices. All studies were performed at 25 °C using
5 nm excitation and emission band widths. Concentrations of
HSA, warfarin, and dansylsarcosine were 2 m M. Amino-acidfluids or L-tryptophan solution (0—50 m l) were added to the
mixture solution (3 ml) of HSA-warfarin or HSA-dansylsarcosine. The fluorophores of warfarin and dansylsarcosine
bound to HSA were excited at 320 and 350 nm, respectively.
Intensities were measured at peak maximum and corrected
for any dilution effects. Although protein concentrations
were maintained at low levels, to minimize inner-filter effect,
the intensities were further corrected using the following
equation;26)
FcFobs antilog[(AexcAem)/2]

where Fobs and Fc are the observed and corrected fluorescence intensities, and Aexc and Aem are the absorbances at excitation and emission wavelengths, respectively.
Ultrafiltration Ultrafiltration experiments were performed using Minicent-10 centrifugal filter devices (Tosoh
Co., Tokyo, Japan). The two compartments were separated
by a cellulose membrane, which had a molecular weight cutoff of 10000 Da. HSA concentration in human serum was adjusted to 500 m M by diluting the serum with phosphate buffer(pH 7.4). Subsequently, an aliquot (200 m l) of the mixture
consisting of the diluted human serum and the radio-labeledprobe (5 m M) was placed in the upper compartment and centrifuged at 3000 rpm for 10 min at 25 °C. The radioactivities
of both solutions before filtration (RAbefore) and after filtration(RAafter) were determined by liquid scintillation counting
after adding scintillation fluid (Nacalai Tesque, Kyoto, Japan)
to scintillation vials. The free fraction of the radio-labeledprobe was calculated as a % ratio of the activities of the solutions before and after filtration using the following equation:

Fig. 1. Effects of Amino-Acid Fluids on the Fluorescence Induced by
HSA-Warfarin (A) and HSA-Dansylsarcosine (B) Interactions
Amino-acid fluids used were PN-twin®-No. 1 (), Fulcaliq® 1 (), Aminotripa®
No. 1 (), Aminofluid® (), Aminoleban® (), Kidmin® (), Amiparen® (), andProteamin 12 X Injection® (). Normal saline solution was used as a control ( ). The
concentration of HSA, warfarin and dansylsarcosine was 2 m M each. Each amino-acidfluid was added to 3 ml of HSA-warfarin or HSA-dansylsarcosine solution. The data
were the mean values of three experiments.

free fraction (%)RAafter/RAbefore100

The effects of amino-acid fluids or L-tryptophan solution
were investigated by the addition of 2 m l of each of these to
the mixture described above. The adsorption of warfarin or
diazepam onto the filtration membrane and the apparatus was
negligible.
RESULTS
Effects of Amino-Acid Fluids on Fluorescence of Site
Marker Fluorescence Probes Bound to HSA Warfarinand dansylsarcosine were used as site marker fluorescenceprobes for monitoring site I and site II of HSA, respectively.2,7,8) The effects of amino-acid fluids on the enhanced
fluorescence of each probe, which is induced by their bindings to HSA, were investigated. As shown in Fig. 1, the fluorescence intensity of the warfarin–HSA complex was not in-March 2005

Fig. 2. Correlation between Fluorescence Intensity (% of Initial Intensity)in the Presence of Amino-Acid Fluids (--- ---) or L-Tryptophan Solutions() and Their L-Tryptophan Contents
50 m l of amino-acid fluids or L-tryptophan solutions was added to 3 ml of HSA-warfarin (A) or HSA-dansylsarcosine (B) solution. Contents of L-tryptophan in amino-acidfluids are indicated in Table 1 and those in L-tryptophan solutions were 0, 0.5, 1.0, 1.5,
2.0, and 2.5 g/l. L-Tryptophan solutions were prepared with nomal saline solution. The
concentration of HSA, warfarin, and dansylsarcosine was 2 m M each. The data were themean values of three experiments.

fluenced by any amino-acid fluids even when larger volumes
were added (volume of 50 m l). However, amino-acid fluids
decreased the fluorescence intensity of dansylsarcosine–HSA
complex in a fluid volume-dependent manner. Among theamino acids included in each fluid, L-tryptophan contents
correlated well with the extent of the fluid’s effects on thedansylsarcosine–HSA interaction (Fig. 2B, closed symbols
and line). The effects of L-tryptophan solutions were also examined to confirm whether L-tryptophan is responsible forthese effects. The plot of the effects (Fig. 2B, open symbols)
coincides well with the correlation curve described by the effects of amino-acid fluids. Thus, it was concluded that Ltryptophan in amino acid fluids plays a major role in thechange in dansylsarcosine–HSA interaction. The other fluids
examined (fluids containing electrolytes, carbohydrates,
and/or trace vitamins) did not show any effects both on warfarin-HSA and on dansylsarcosine–HSA interactions (datanot shown).
Effects of Amino-Acid Fluids on Bindings of Drugs to
HSA in Human Serum The binding of warfarin and diazepam to HSA in human serum was examined using ultrafiltration techniques. Warfarin and diazepam are drugs thatbind specifically to site I and site II of HSA, respectively.1,2)
As shown in Fig. 3, amino-acid fluids and L-tryptophan solution selectively increased the free fraction of diazepam without any effect on warfarin binding. The potency of the effects
on diazepam binding correlated well with the L-tryptophan
contents in the amino-acid fluids (Fig. 3B, closed symbols
and line) and in the L-tryptophan solution (Fig. 3B, open
symbols). These results indicated that L-tryptophan in theamino-acid fluids inhibits the drug binding to site II of HSA
in human serum.

551

Fig. 3. Effects of Amino-Acid Fluids (--- ---) or L-Tryptophan Solutions() on the Free Fraction of [14C]Warfarin (A) and [14C]Diazepam (B) in
Human Serum
Normal saline solution was used as a control (L-tryptophan contents; 0 g/l). The concentration of HSA in human serum was adjusted to 500 m M, those of [14C]warfarin and
14[ C]diazepam were 5 m M. 2 m l of amino-acid fluids were added to 200 m l of [14C]warfarin- or [14C]diazepam-human plasma mixture solution. Results are presented as themeanS.D. of three independent experiments.

DISCUSSION
Parenteral nutrition is now widely used for the treatmentof malnutrition.18—20) Among the components of parenteralnutrition, fluids including amino acids, electrolytes, carbohydrates, and/or trace vitamins (Table 1) were investigated withrespect to their effects on protein binding.
The fluorescence technique, which is a useful tool for
rapid screening of the factors that affect the ligand interaction with each binding site of HSA,7,8,27,28) showed thatamino-acid fluids had specific effects on only the interactionof dansylsarcosine with site II of HSA without any effects on
the interaction of warfarin with site I (Fig. 1). Althoughamino-acid fluids also contain electrolytes, carbohydrates
and/or trace vitamins, fluids containing electrolytes, carbohydrates, or trace vitamins did not show any effects (data notshown). Thus, amino acid(s) contained in fluids might affectthe ligand interaction with site II of HSA.
Site II specific effects were also observed in drug binding
to human serum. The ultrafiltration results indicated that theamino-acid fluids specifically displaced the site II drug, diazepam, without any effects on the binding of the site I drug,
warfarin (Fig. 3). Since warfarin and diazepam specificallybind to HSA in human serum,29—32) these findings could be
explained by the specific inhibition of the binding to site II of
HSA, which is consistent with the results of the fluorescence
technique. Furthermore, the site II specific effects of aminoacid fluids, observed by both fluorescence and ultrafiltration
techniques, were confirmed to be only due to the L-tryptophan contents in amino-acid fluids. This was based on thecomparison of the results between the effects of amino-acidfluids and those of L-tryptophan solutions. Plots demonstrating the effects of L-tryptophan solutions fit the correlation
curve of the effects between amino-acid fluids and their Ltryptophan contents (Figs. 2, 3).

552
L-Tryptophan has been known to be the only aminoacid that binds to one high affinity binding site of HSA, site
II, with an affinity constant of 1—6.2104 M1 at 25—
37 °C.33—35) Kragh-Hansen36) reported that diazepam and Ltryptophan compete for a common high affinity binding siteon HSA. Therefore, our present findings suggest that the reduction in binding of diazepam to site II by amino-acid fluids
is due to the competitive displacement by L-tryptophan in thefluids. The affinity constant of L-tryptophan is lower than thatof commonly used site II drugs (105—106 M1).1,2) The increased concentration of amino acid upon infusion of aminoacid fluid was reported to be closely correlated to their infusion rate in healthy volunteers (increased concentration1.46infusion rate18.27, r 20.92).37) According tothis correlation, the concentration of L-tryptophan might increase by 12 m M (for Kidmin®) higher than the concentration
prior to infusion (about 40 m M), whereas, in our experiments,
the final concentrations of increased L-tryptophan in humanserum are 13 m M (for PN-twin-No. 1®)—122 m M (for Kidmin®). Thus, in vivo studies on the effect of co-administeredamino-acid fluids on drug binding to site II should be undertaken in the future. This may be especially important in patients with certain disease states in which albumin concentration is decreased (e.g. malnutrition, liver dysfunction, nephrosis, GI losses, shock, edema).1) Monitoring of free drug concentrations may be useful for these and other patients inwhom parenteral nutrition with amino-acid fluids is used,particularly for those drugs that are bound to site II on HSA.
In conclusion, L-tryptophan in amino-acid fluids competitively displaces the drug bound to site II on HSA. The present findings are very useful for evaluating the pharmacokinetics and pharmacological effects of therapeutic drugs inpatients on parenteral nutrition with amino-acid fluids.

Acknowledgments This research was supported in partby a Grant-in-Aid (#16922103) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

Vol. 28, No. 3

7)
8)
9)
10)
11)
12)
13)
14)
15)
16)
17)
18)
19)
20)
21)
22)
23)
24)
25)
26)
27)
28)

29)
30)

REFERENCES

31)

1)

32)

Peters T., Jr., “All about Albumin: Biochemistry, Genetics, and Medical Applications,” Academic Press, San Diego, 1996.
2) Kragh-Hansen U., Chuang V. T., Otagiri M., Biol. Pharm. Bull., 25,
695—704 (2002).
3) Carter D. C., Ho J. X., Adv. Protein Chem., 45, 153—201 (1994).
4) Sellers E. M., Koch-Weser J., “Albumin Structure, Function and Uses,”ed. by Rosenoer V. M., Oratz M., Rothschild M. A., Pergamon Press,
Oxford, 1977, pp. 159—182.
5) Vallner J. J., J. Pharm. Sci., 66, 447—465 (1977).
6) McMenamy R. P., “Albumin Structure, Function and Uses,” ed. byRosenoer V. M., Oratz M., Rothschild M. A., Pergamon Press, Oxford,

33)
34)
35)
36)
37)

1977, pp. 143—158.
Sudlow G., Birkett D. J., Wade D. N., Mol. Pharmacol., 11, 824—832(1975).
Sudlow G., Birkett D. J., Wade D. N., Mol. Pharmacol., 12, 1052—
1061 (1976).
Sjöholm I., Ekman B., Kober A., Ljungstedt-Pahlman I., Seiving B.,
Sjödin T., Mol. Pharmacol., 16, 767—777 (1979).
He X. M., Carter D. C., Nature (London), 358, 209—215 (1992).
Petitpas I., Bhattacharya A. A., Twine S., East M., Curry S., J. Biol.
Chem., 276, 22804—22809 (2001).
Bos O. J., Remijn J. P., Fischer M. J., Wilting J., Janssen L. H.,
Biochem. Pharmacol., 37, 3905—3909 (1988).
Bos O. J., Fischer M. J., Wilting J., Janssen L. H., Biochem. Pharmacol., 38, 1979—1984 (1989).
Dockal M., Carter D. C., Ruker F., J. Biol. Chem., 274, 29303—29310(1999).
Dockal M., Chang M., Carter D. C., Ruker F., Protein Sci., 9, 1455—
1465 (2000).
Wilmore D. W., Dudrick S. J., JAMA, 203, 860—864 (1968).
Murphy A., Scott A., Hosp. Pharm., 7, 146—154 (2000).
ASPEN Board of Directors and The Clinical Guidelines Task Force,
JPEN, 26, 1SA—138SA (2002).
Worthington P., Gilbert K. A., Wagner B. A., AACN Clin. Issues., 11,
559—579 (2000).
National Advisory Group on Standards of Practice Guidelines for Parenteral Nutrition, JPEN, 22, 49—66 (1998).
Walter-Sack I., de Vries J. X., Rudi J., Conradi R., Kohlmeier M., Kohl
B., Weber E., Eur. J. Clin. Pharmacol., 42, 71—75 (1992).
Sangrador G., Sanchez-Alcaraz A., Ubeda R., Ibanez P., J. Clin.
Pharm. Ther., 15, 273—278 (1990).
Pantuck E. J., Weissman C., Pantuck C. B., Lee Y. J., Anesth. Analg.,69, 727—731 (1989).
Burgess P., Hall R. I., Bateman D. N., Johnston I. D., JPEN, 11, 540—
543 (1987).
Bailey D. N., Briggs J. R., Ther. Drug Monit., 26, 31—34 (2004).
Lackowicz J. R., “Principles of Fluorescence Spectroscopy,” Plenum
Press, New York, 1983.
Yamasaki K., Maruyama T., Kragh-Hansen U., Otagiri M., Biochim.
Biophys. Acta, 1295, 147—157 (1996).
Yamasaki K., Maruyama T., Yoshimoto K., Tsutsumi Y., Narazaki R.,
Fukuhara A., Kragh-Hansen U., Otagiri M., Biochim. Biophys. Acta,
1432, 313—323 (1999).
Yacobi A., Udall J. A., Levy G., Clin. Pharmacol. Ther., 19, 552—558(1976).
Piroli R. J., Passananti G. T., Shively C. A., Vesell E. S., Clin. Pharmacol. Ther., 30, 810—816 (1981).
Martyn J. A., Abernethy D. R., Greenblatt D. J., Clin. Pharmacol.
Ther., 35, 535—539 (1984).
Grossman S. H., Davis D., Kitchell B. B., Shand D. G., Routledge P.
A., Clin. Pharmacol. Ther., 31, 350—357 (1982).
Hage D. S., J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 768,
3—30 (2002).
Yang J., Hage D. S., J. Chromatogr. A, 725, 273—285 (1996).
Sollenne N. P., Wu H. L., Means G. E., Arch. Biochem. Biophys., 207,
264—269 (1981).
Kragh-Hansen U., Biochem. J., 209, 135—142 (1983).
Berard M. P., Hankard R., Cynober L., Clin. Nutr., 20, 407—414(2001).